Precise timing of sperm activation ensures the greatest likelihood of fertilization. Precision in Caenorhabditis elegans sperm activation is ensured by external signaling, which induces the spherical spermatid to reorganize and extend a pseudopod for motility. Spermatid activation, also called spermiogenesis, is prevented from occurring prematurely by the activity of SPE-6 and perhaps other proteins, termed "the brake model." Here, we identify the spe-47 gene from the hc198 mutation that causes premature spermiogenesis. The mutation was isolated in a suppressor screen of spe-27(it132ts), which normally renders worms sterile, due to defective transduction of the activation signal. In a spe-27(+) background, spe-47(hc198) causes a temperature-sensitive reduction of fertility, and in addition to premature spermiogenesis, many mutant sperm fail to activate altogether. The hc198 mutation is semidominant, inducing a more severe loss of fertility than do null alleles generated by CRISPR-associated protein 9 (Cas9) technology. The hc198 mutation affects an major sperm protein (MSP) domain, altering a conserved amino acid residue in a b-strand that mediates MSP-MSP dimerization. Both N-and C-terminal SPE-47 reporters associate with the forming fibrous body (FB)-membranous organelle, a specialized sperm organelle that packages MSP and other components during spermatogenesis. Once the FB is fully formed, the SPE-47 reporters dissociate and disappear. SPE-47 reporter localization is not altered by either the hc198 mutation or a C-terminal truncation deleting the MSP domain. The disappearance of SPE-47 reporters prior to the formation of spermatids requires a reevaluation of the brake model for prevention of premature spermatid activation.

To acquire and maintain directed cell motility, Caenorhabditis elegans sperm must undergo extensive, regulated cellular remodeling, in the absence of new transcription or translation. To regulate sperm function, nematode sperm employ large numbers of protein kinases and phosphatases, including SPE-6, a member of C. elegans’ highly expanded casein kinase 1 superfamily. SPE-6 functions during multiple steps of spermatogenesis, including functioning as a “brake” to prevent premature sperm activation in the absence of normal extracellular signals. Here we describe the sub-cellular localization patterns of SPE-6 during wildtype C. elegans sperm development and in various sperm activation mutants. While other members of the sperm activation pathway associate with the plasma membrane or localize to the sperm’s membranous organelles, SPE-6 surrounds the chromatin mass of unactivated sperm. During sperm activation by either of two semiautonomous signaling pathways, SPE-6 redistributes to the front, central region of the sperm’s pseudopod. When disrupted by reduction-of-function alleles, SPE-6 protein is either diminished in a temperature-sensitive manner (hc187) or is mis-localized in a stage-specific manner (hc163). During the multistep process of sperm activation, SPE-6 is released from its perinuclear location after the spike stage in a process that does not require fusion of membranous organelles with the plasma membrane. After activation, spermatozoa exhibit variable proportions of perinuclear and pseudopod-localized SPE-6, depending on their location within the female reproductive tract. These findings provide new insights regarding SPE-6’s role in sperm activation and suggest that extracellular signals during sperm migration may further modulate SPE-6 localization and function.

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The activation of C. elegans spermatids to crawling spermatozoa is affected by a number of genes including spe-47. Here, we investigate a paralog to spe-47: spe-50, which has a highly conserved sequence and expression, but which is not functionally redundant to spe-47. Phylogenetic analysis indicates that the duplication event that produced the paralogs occurred prior to the radiation of the Caenorhabditis species included in the analysis, allowing a long period for the paralogs to diverge in function. Furthermore, we observed that knockout mutations in both genes, either alone or together, have little effect on sperm function. However, hermaphrodites harboring both knockout mutations combined with a third mutation in the him-8 gene are nearly self-sterile due to a sperm defect, even though they have numerous apparently normal sperm within their spermathecae. We suggest that the sperm in these triple mutants are defective in fusing with oocytes, and that the effect of the him-8 mutation is unclear but likely due to its direct or indirect effect on local chromatin structure and function.